Epidemiologically relevant phthalates affect human endometrial cells in vitro through cell specific gene expression changes related to the cytoskeleton and mitochondria.

Phthalates are endocrine disrupting chemicals (EDCs) found in common consumer products such as soft plastics and cosmetics. Although the knowledge regarding the adverse effects of phthalates on female fertility are accumulating, information on the hormone sensitive endometrium is still scarce. Here, we studied the effects of phthalates on endometrial cell proliferation and gene expression. Human endometrial primary epithelial and stromal cells were isolated from healthy fertile-aged women (n=3), and were compared to endometrial cell lines T-HESC and Ishikawa. Three different epidemiologically relevant phthalate mixtures were used, defined by urine samples in the Midlife Women Health Study (MWHS) cohort. Mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was used as a single phthalate control. Cells were harvested for proliferation testing and transcriptomic analyses after 24 h exposure. Even though all cell models responded differently to the phthalate exposures, many overlapping differentially expressed genes (DEGs, FDR<0.1), related to cell adhesion, cytoskeleton and mitochondria were found in all cell types. The qPCR analysis confirmed that MEHHP significantly affected cell adhesion gene vinculin (VCL) and NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7), important for oxidative phosphorylation. Benchmark dose modelling showed that MEHHP had significant concentration-dependent effects on cytoskeleton gene actin-beta (ACTB). In conclusion, short 24 h phthalate exposures significantly altered gene expression cell-specifically in human endometrial cells, with six shared DEGs. The mixture effects were similar to those of MEHHP, suggesting MEHHP could be the main driver in the mixture. Impact of phthalate exposures on endometrial functions including receptivity should be addressed.

Endocrine disrupting chemicals interfere with decidualization of human primary endometrial stromal cells in vitro.

Multiple studies have shown associations between exposure to endocrine disrupting chemicals (EDCs) and reduced fertility in women. However, little is known about the target organs of chemical disruption of female fertility. Here, we focus on the hormone-sensitive uterine lining, the endometrium, as a potential target. Decidualization is the morphological and functional change that endometrial stromal cells undergo to support endometrial receptivity, which is crucial for successful implantation, placentation, and pregnancy. We investigated the effect of nine selected EDCs on primary human endometrial stromal cell decidualization in vitro. The cells were exposed to a decidualization-inducing mixture in the presence or absence of 1 µM of nine different EDCs for nine days. Extent of decidualization was assessed by measuring the activity of cAMP dependent protein kinase, Rho-associated coiled-coil containing protein kinase, and protein kinase B in lysates using photoluminescent probes, and secretion of prolactin into the media by using ELISA. Decidualization-inducing mixture upregulated activity of protein kinases and prolactin secretion in cells derived from all women. Of the tested chemicals, dichlorodiphenyldichloroethylene (p,p'-DDE), hexachlorobenzene (HCB) and perfluorooctanesulfonic acid (PFOS) significantly reduced decidualization as judged by the kinase markers and prolactin secretion. In addition, bisphenol A (BPA) reduced prolactin secretion but did not significantly affect activity of the kinases. None of the EDCs was cytotoxic, based on the assessment of total protein content or activity of the viability marker casein kinase 2 in lysates. These results indicate that EDCs commonly present in the blood circulation of reproductive-aged women can reduce decidualization of human endometrial stromal cells in vitro. Future studies should focus on detailed hazard assessment to define possible risks of EDC exposure to endometrial dysfunction and implantation failure in women.

Vasoactive intestinal peptide is upregulated in women with endometriosis and chronic pelvic pain.

PROBLEM: Chronic pelvic pain (CPP) causes compromised the quality of life in women with endometriosis and is often attributed to local inflammation and ingrowth of nerve fibers. In this pilot study, we aimed to investigate whether the inflammation-related vasoactive intestinal peptide (VIP) and interleukin (IL)-6 were increased in affected patients. METHOD OF STUDY: Endometrial and endometriotic tissue biopsy specimens, and serum and peritoneal fluid (PF) samples, were obtained from 85 endometriosis patients and 53 controls. VIP and IL-6 analysis and measurement of microvessel density in tissue were performed using immunohistochemistry, Western blotting, RT-qPCR, and ELISA. RESULTS: Compared with controls, VIP transcript and protein levels were increased in endometrium from endometriosis patients and further elevated in patients with CPP. In addition, microvessel density, a measurement of angiogenic activity, was increased in the endometrium and in endometriosis lesions in the same subset of patients. Serum and PF levels of VIP and IL-6 were higher in women with endometriosis and CPP compared with endometriosis patients who reported no chronic pain. CONCLUSION: Vasoactive intestinal peptide is upregulated in endometriosis patients reporting chronic pain. Increased microvessel density in tissue and peritoneal fluid concentrations of IL-6 indicate an elevated inflammation in the pelvic microenvironment of these patients.

Physical activity, fatness, educational level and snuff consumption as determinants of semen quality: findings of the ActiART study.

In this study, the association between physical activity and other potential determinants, objectively measured by accelerometry, was examined. Sixty-two men attending an infertility clinic participated in the study. Obese men (body mass index ≥ 30) and those with a waist circumference 102 cm or more had lower semen volume than the other men (P < 0.05). Higher values in sperm parameters were observed in participants who completed university studies and those who did not consume snuff, compared with the other participants (P < 0.05). Finally, men who spent an average number of 10 min-bouts of moderate-to-vigorous physical activity had significantly better semen quality than those who engaged in low or high numbers of bouts of activity (P < 0.05). No associations were found for sedentary or moderate-to-vigorous physical activity time when it was not sustained over 10 min, i.e. not in bouts. Men who have average levels of physical activity over sustained periods of 10 min are likely to have better semen quality than men who engage in low or high levels of such activity. Similarly, high levels of total and central adiposity, low educational level and snuff consumption are negatively related to semen quality.

Bisphenol A affects human endometrial endothelial cell angiogenic activity in vitro.

The widespread Bisphenol A (BPA) is classified as an endocrine-disrupting chemical (EDC) with estrogenic properties. Human endometrial endothelial cells (HEECs) play a key role in the endometrial angiogenesis that is under the control of estradiol. The hypothesis was that BPA may affect endometrial angiogenesis by disturbing some functional properties of the HEEC. To study this, primary HEECs were exposed to environmentally relevant doses of BPA. The HEECs were co-cultured with primary endometrial stromal cells to create conditions as similar to the in vivo situation as possible. The effects of BPA were evaluated by proliferation and viability assays, tube-formation assays, quantitative PCRs, Western blots and ELISAs. BPA slightly increased HEEC tube formation and VEGF-D protein expression compared with vehicle, without affecting HEEC viability or proliferation. Bisphenol A thus caused changes in HEEC activities in vitro, and may therefore have disturbing effects on endometrial angiogenesis.

Subcutaneous fat tissue for diagnosis and studies of systemic amyloidosis.

The systemic amyloidoses comprise a biochemically heterogeneous group of potentially lethal disorders. An early and precise diagnosis is crucial for the treatment and prognosis. Subcutaneous fat biopsy is a simple and safe method to obtain a diagnosis of systemic amyloidosis and the material can be used for exact determination of amyloid type. A method is described for immunochemical typing of the amyloid based on Western blot analysis combined with specific amyloid fibril protein antibodies.